HBV Banana Vaccines
Despite the availability of an efficacious HBV vaccine since 1982, Hepatitis B continues to be a considerable cause of morbidity and mortality internationally (5). It is speculated that over 350 million individuals, or approximately 5% of the global population, are chronic carriers of the virus and of these 350 million, 70-100 million will die due to complications of liver cirrhosis and/or heptacellular carcinoma (6). In the USA, more than 300,000 citizens are postulated to contract HBV annually, despite the availability of the vaccine and the CDC's recommendation to have all children immunized at birth. Hepatitis B virus is spread through the exchange of bodily fluids (i.e. through sexual intercourse or through contaminated needles) and is 50-100 times more infectious than HIV. HBV is the single most common cause of persistent viremia in humans internationally and the prognosis of all those who contract HBV is generally pretty bleak as the virus is incurable (5,6). Why then is the vaccine not in wider circulation? The main barrier is cost. The current vaccine in circulation is administered over a 6 month period in a series of three intramuscular injections. The injections themselves must be delivered by medically trained personnel and the vaccine must remain refrigerated prior to inoculation (5). Also contributing to the high cost of HBV immunization is the necessity of a sterilized syringe upon every inoculation. With all of these factors considered and ignoring other potential costs (i.e. transportation of the vaccine), the cost of immunizing a single individual against HBV exceeds $100, making an international HBV eradication campaign a tragically satirical notion (5). These daunting figures have encouraged researchers and benefactors alike to seek alternative HBV vaccines. One of the more promising prospects is the development of edible vaccines. Edible vaccines are plant-derived, orally delivered vaccines and though the idea may sound futuristic, edible vaccines have been in production since the mid 1990s, though as to date no edible vaccines have yet made it out of clinical trials into practice (3,6). Edible Vaccines One of the first professionals to explore the idea of edible vaccines was Dr. Charles Arntzen, a regent professor of infectious disease and vaccinology at Arizona State University, who has been pioneering edible vaccine research since the mid 1990s (3). Arntzen believes that plant derived vaccines could one day front-line eradication campaigns for a myriad of infectious diseases as they offer numerous advantages over their existing counterparts. For one thing, edible vaccines bypass the need for sterile syringes, trained medical personnel, refrigeration and multiple injections, significantly mitigating the exorbitant costs associated with many current vaccines. It has been postulated that ingested vaccines may actually offer superior protection against pathogens that invade te body via the gastrointestinal tract (i.e. cholera, polio, rotavirus) as they "mimic the natural route of infection (3)". Individuals are also likely to be more willing to eat a banana (or other transgenic fruit) than receive an injection. So far, Dr. Arntzen has conducted clinical trials with genetically engineer potatoes fortified with Norwalk virus surface antigen with promising results. Dr. Arntzen is also fronting efforts to develop an edible cholera vaccine. Due to the astounding degree of morbidity and mortality attributable to HBV, it did not take researchers long to exploit this new technology and apply it to the development of an edible HBV vaccine. In 1999, Boyce Thompson Institute for Plant Research Incorporated (BTI), a non-profit Cornell research affiliate in Buffalo, New York, began the first clinical trails ever conducted testing the efficacy of an edible HBV vaccine (5). For their clinical trials, the BTI employed transgenic Solanum tuberosum ''(potatoes) fortified with a gene encoding hepatitis B surface antigen (HBsAg). In 2005, Dr. Yasmin Thanavala and associates within the RCPI (Royal College of Physicians of Ireland) published the results of clinical trial evaluating the immunogenicity their own transgenic, HBV potatoes. In Dr. Thanavala's double-blind, placebo-controlled clinical trial, more than 40 health care workers who had been previously vaccinated against HBV volunteered to eat multiple servings of raw transgenic or control potatoes (6). Volunteers were randomly partitioned into 3 groups prior to the commencement of the experiment: Group A received 3 servings/doses of uncooked, transgenic potato, group B received 2 servings/does of uncooked, transgenic potato and group C received raw, non-transgenic potatoes. 10 of the 16 participants in group A saw a substantial increase in serum anti-HBsAg titers (i.e. the concentration of circulating antibody specific to HBV surface antigen) while 9 out of the 17 members of group B saw a notable increase in serum anti-HBsAg titers. No participants within group C developed an increased anti-HBsAg titer. These results, while far from perfect, show that edible vaccines, with further refinement, could be a viable alternative to the immunization techniques used today in future eradication campaigns (6). Dr. Yasmin Thanavala however feels that the transgenic HBV potato currently undergoing clinical trials is far from ideal the ideal vaccine vector, as few people eat potatoes raw. According to Dr. Thanavala, the end goal would be to stably introduce the HBV surface antigen into an agricultural product consumed world wide that is preferably eaten raw, criteria that bananas met (5,6). Since the conclusion and publication of Dr. Thanavala's clinical trial in 2005 numerous researchers have joined the edible HBV vaccine crusade and so far, transgenic tomatoes, potatoes, bananas, carrots and lettuces all fortified with the HBV surface antigen have been engineered (though few of these products have reached clinical trials). Dr. Arntzen himself has participated in this international effort. By 2000, Dr. Arntzen had engineered a transgenic HBV tomato that expressed such a stable variant of the HBV surface antigen, it was able to elicit an immune response even after the tomato had been processed or dried (4). According to Arntzen, his 30 original transgenic tomato plants, which typically yield 15lbs of tomatoes weekly "were enough to produce thousands of doses of effective vaccine at 1 cent per 4 doses (4)". Arntzen's tomatoes and other such edible vaccines are unlikely to replace their older, more costly counterparts in the US anytime soon however as the FDA is yet to formulate any sort of licensing guidelines for edible vaccines. Synthesis and Method of Action While there are a variety of hypothetical ways one could engineer a transgenic organism expressing the HBV surface antigen, only one sample technique will be discussed in detail. First, Reverse Transcription PCR (RT-PCR) is used to amplify viral mRNA encoding the HBV surface antigen protein, resulting in the accumulation of double stranded DNA encoding this viral genomic protein (1). A single HBsAg DNA gene, an antibiotic resistance gene and a reporter gene (e.g. GUS) are all inserted into plasmid vectors using restriction enzymes. These newly synthesized plasmid vectors are then introduced into ''Agrobacterium tumefaciens bacteria using either transduction or transformation. These bacteria are then grown on a growth medium containing the antibiotic that the designed plasmid confers resistance to so that only bacteria that have acquired the plasmid will be isolated. These transformed bacteria are then allowed to temporarily infect the agricultural crop of interest (in this case bananas) (1,6). The treated crops are then cured of the bacterial infection and allowed to grow to maturity. Upon maturity, plants that are determined to contain the insert HBV surface antigen gene and be properly expressing the protein clonally propagated to generate a second generation of transgenic organisms. Recent research has discovered that these transgenic HBsAg proteins fold spontaneously within plant cells and that no further modification is needed to generate immunity in human subjects (1,5). Crops expressing the HbsAg protein or food products containing these crops could then hypothetically be distributed to interested parties. How could an ingested, transgenic banana provide immunity to a hematogenous hepatic disease you may ask? When a transgenic banana (or other food product) containing the HBV surface antigen is ingested, the HBV viral antigen will be encountered by microfold cells (M-cells) within the lumen of the small intestine. M-cells will recognize the antigen as foreign and possibly pathogenic, endocytosizing the protein and presenting it to the dendritic, antigen-presenting cells and lymphocytes adjacent to their basolateral membranes (1). These cells work in tandem to sensitize and activate B-lymphocytes so that they will secrete antibodies specific to HBsAg, thereby providing the individual with future immunity to the pathogen. Once one is found to be producing these antibodies, they are said to have been vaccinated against Hepatitis B virus. Possible Disadvantages of the Banana HBV Vaccine Unfortunately, a world where one could administer a transgenic banana to a squealing infant, simultaneously nourishing and immunizing them against HBV exists in a utopian future many years away. Even more tragic is that many of the barriers preventing this from becoming a reality have their roots in capitalism and money mongering. Cultivating any sort of crop commercially typically requires extensive amounts of land and banana plantations in particular require a great deal of space and maintenance. It has thus been extremely difficult to find plantations willing to grow bananas that could not be sold in the commercial, agricultural market (1). As the amount of HBsAg protein actually within a particular banana currently varies with weight and ripeness, administration of the edible vaccines would require trained medical professionals and possibly expensive laboratory equipment to ensure a relatively consistent dosage was administered to each patient (1). It is possible that these difficulties could be overcome with further refinement of the technique or the implementation of some sort of standardized processing procedure (i.e. harvesting the bananas at a specific point of ripeness and then processing them into banana chips of a certain weight and then distributing the banana chips with instructions like "2 chips per patient"). Only time will tell what the future holds for edible vaccines. References 1.Prezi: The Banana Vaccine 2.Science Daily 3.Dr. Charles Arntzen and Edible Vaccines 4.The Guardian: Vaccine in GM fruit could wipe out hepatitis B 5.A Needle or a Banana? 6.Immunogenicity in humans of an edible vaccine for hepatitis B